Induced expression of genetic material, for example DNA sequences, in living cells is the foundation for molecular genetics and molecular biology. In this process, genetic material encoding for example for genes, is artificially introduced into the nucleus of a cell, so that the cell generates the product of the genes in the form of non-native or modified proteins, as is achieved by genetic engineering. In microbiology, the method by which one or more particular genes is altered in a recipient cell is referred to as transformation. In eukaryotic biology this method is sometimes referred to as transfection, or the introduction of a foreign cloned gene or cDNA into the eukaryotic genome. Viral or plasmid vectors are the vehicle for the introduction of DNA sequences.
Many methods have been employed to introduce genetic material into the nucleus or cytoplasm of living cells. These methods have limitations and require the conveyance of plasmid cDNA across plasma membranes, which are the heterogeneous bilayers of lipid molecules found on all cells. A well-known method to introduce genetic material into the nucleus or cytoplasm of living cells is electroporation, which relies on dielectric breakdown of the membrane producing gaps of up to 120 nanometers (nm) in diameter (metastable aqueous pores) in the membrane through which genetic material enters the cell through electrodiffusion. Another method is the use of transfection reagents including lipid or fat-based reagents (i.e., lipofectamine) which are essentially detergents that associate with DNA, thereby permitting the DNA to pass though the plasma membrane.
Biolistic transfection is yet another method, and involves a “gene gun” that fires the genetic material coupled to gold nanoparticles at high pressure through the plasma membrane. Mechanical injection is yet another method that typically uses glass needles that physically puncture the plasma membrane to deliver the genetic material using hydrostatic pressure injection directly into the nucleus.
Efficiencies of these methods have been found to have limitations, particularly due to toxicity, injury, or death to the cells. The transfection efficiency of such methods typically is very low or variable, depending upon the method and the cell type, and is related to the loss of viability of the treated cells, or the inability of the method to get the genetic material through the cell membrane. For example, transfection efficiency of the gene gun to treat a variety of different cell lines was observed to be successful with only about 1% to 4% of treated cells. Lipofection yields successful recombinants in only about 10% to 20% of treated cells. Polycationic lipid reagents generally do not exceed 40% efficiency. Most importantly, certain cell types are not amenable to any method, including certain types of immune system cells, human stem cells, muscle cells, nerve cells, and other cell types that do not divide and are therefore maintained in culture only as primary cells.
A more biological approach relies on virus-mediated transfection for introduction of genetic material into cells. This approach utilizes specific engineered viral vectors derived from strains such as adenovirus, sindbis virus, retrovirus, baculovirus or lentivirus, etc. to infect the cells. The viral genomes are engineered to carry the genetic sequences of interest, and are consequently a relatively time-consuming and labor-intensive method compared to mechanical means of introducing genetic material. This approach has a significant methodological limitation in that the time delay or lag to obtain protein expression depends not only on the efficiency of the cell to transcribe and translate the genetic material, but also on the infection efficiency of the virus. In addition, use of live virus requires special laboratory safety standard conditions, i.e., biohazard level 2.
Infection efficiency depends on many variables, and viral-mediated engineering is characterized by a lag of at least a day or several days to observe protein expression in cell populations. Furthermore, infection efficiency is cell-type specific, i.e., particular viruses do not infect certain cells.
There remains a need for a rapid, efficient, and universal method for introducing material such as genetic material into any type of cell, including cell types that have heretofore been difficult or impossible to transfect with any commercially available method.